Anti-Inflammatory Effect of Three Isolated Compounds of Physalis alkekengi var. franchetii (PAF) in Lipopolysaccharide-Activated RAW 264.7 Cells

(1) Background: Three isolated compounds from Physalis alkekengi var. franchetii (PAF) have been investigated to possess a variety of biological activities. Their structures were elucidated by spectroscopic analysis (Ultraviolet (UV), High-resolution electrospray mass spectrometry (HR-ESI-Ms), and their anti-inflammatory effects were evaluated in vitro; (2) Methods: To investigate the mechanisms of action of PAF extracts and their isolated compounds, their anti-inflammatory effects were assessed in RAW 264.7 macrophages stimulated by lipopolysaccharide (LPS). RAW 264.7 cells were treated with different concentrations of Physalis alkekengi var. franchetii three isolated compounds of PAF for 30 min prior to stimulation with or without LPS for the indicated times. The inflammatory cytokines, interleukin (IL)-1β and tumor necrosis factor (TNF)-α were determined using reverse transcription-polymerase chain (RT-PCR); (3) Results Treatment of RAW 264.7 cells with LPS alone resulted in significant increases in inflammatory cytokine production as compared to the control group (p < 0.001). However, with the treatment of isophysalin B 100 μg/mL, there was a significant decrease in the mRNA expression levels of TNF-α in LPS-stimulated raw 264.7 cells (p < 0.001). With treatment of physalin 1–100 μg/mL, there was a markedly decrease in the mRNA expression levels of TNF-α in LPS stimulated raw 264.7 (p < 0.05). Moreover, TNF-α mRNA (p < 0.05) and IL-1β mRNA (p < 0.001) mRNA levels were significantly suppressed after treatment with 3′,7-dimethylquercetin in LPS stimulated Raw 264.7 cells; (4) Conclusions: These findings suggest that three isolated compounds from can suppress inflammatory responses in LPS stimulated macrophage.


Introduction
The fruit calyxes of Physalis alkekengi L. var. franchetii (PAF) (Mast.) Makino (Solanaceae) have been widely used as traditional and indigenous medicines for therapy for asthma, cough, sore throat, eczema, or urinary problems [1]. There have been phytochemical reports on a variety of types of ingredients from P. alkekengi var. franchetii (PAF) including physalins, steroids, glycosids, flavonoids, and alkaloids [2]. The extracts and some isolates of this plant have been adopted for the treatment of anti-inflammation, cough, antibacterial [3], antipyretic, antioxidant [3], urinary problem, and antiasthma. The latest pharmacological studies proved its uses in fork medicines, however, the molecular mechanisms of purified compounds remained unclear and were worthy of further exploration. Furthermore, there is little scientific evidence regarding the efficacy of PAF-active compounds in inflammation.
In order to examine their potential anti-inflammatory effects of bioactive compounds from PAF, we isolated three compounds from PAF, identified as 3,7-dimethylquercetin, physalin B, and isophysalin B. The known flavonoid, 3,7-dimethylquercetin was, for the first time, isolated as constituents of PAF and has never been isolated from the genus Physalis. The phytochemical and pharmacological activity of three compounds was investigated in the present study.
Based on the highly acclaimed properties of PAF, the present study aimed to evaluate anti-inflammatory (suppression of TNF-α and IL-1β production in LPS-stimulated RAW264.7 cells) activities of ethanol or hexane fraction and their isolated sub-fractions, physalin B, isophysalin B, and 3,7-dimethylquercetin of P. alkekengi var. franchetii using the RT-PCR.

Cell Culture
The RAW264.7 mouse macrophage cell line has been used extensively to carry out in vitro screens for anti-inflammatory candidate agents [5]. The RAW264.7 cell line response is considered to reflect the potential human de novo response and was used to evaluate the isolated three compounds from PAF for bioactivity and to predict their potential effect in vivo or on primary cells. Mouse macrophage RAW 264.7 cells were obtained from the Korean Cell Line Bank (Seoul, Korea) were routinely kept in a Dulbeco's Modified Eagle's Medium supplemented with 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), 100 U/mL penicillin G and 100 µg/mL streptomycin at 37 • C in a 5% CO 2 air incubator under standard conditions.

LPS-Induced Inflammation and RT-PCR Analysis
RAW 264.7 cells in 6 well plates (1 × 10 5 cells/well, 500 µL medium/well) were pretreated with various concentrations of P. alkekengi var. franchetii compounds (1-100 µg/mL) for 4 h prior to incubation for 2 h at 37 • C in an incubator with 5% CO 2 , with or without 1 µg/µL LPS. Total RNA was extracted from the RAW 264. The final PCR products were separated with 1.2% agarose gels, stained with ethidium bromide. The band intensities were measured by densitometric analysis ImageMaster TotalLab (Amersham Pharmacia Biotech, Uppsala, Sweden) and were expressed relative to the intensity of the GAPDH band.

Quantitative Real-Time PCR (qPCR)
Total RNA was isolated from transiently transfected cells (TRIzol reagent, Invitrogen, CA, USA), reverse transcribed (Superscript III, Invitrogen, CA, USA), and subjected to quantitative PCR analysis using SYBER green master mix (Invitrogen, CA, USA). qPCR was performed with ABI PRISM 7700 Sequence Detection System Instrument and software (Applied Biosystems, Foster City, CA, USA), using the manufacturer's recommended conditions. The comparative threshold cycle (Ct) method was used to calculate the amplification factor, and the relative number of targets was normalized to GAPDH levels in parallel reactions. The primer sequences are described in Table 1.

Statistical Analysis
The values of the experimental results were expressed as the mean ± S.E.M. Statistical analysis was used with SPSS 25.0 software (SPSS 25 Inc., Chicago, IL, USA). Differences among groups were analyzed using one-way ANOVA and LSD post hoc test. A p-value of less than 0.05 was considered statistically significant. Graph generation was followed with Graphpad Prism 6.0 version software.

Effects of Compounds from Ethanol Extract in Lipopolysaccharide (LPS) Stimulated RAW 264.7 Cells
The expression level of TNF-α and IL-1β mRNA in the LPS stimulated RAW 264.7 cells was measured by an RT-PCR (Figure 2A-D). As shown in Figure 2A, treatment with LPS alone of RAW 264.7 cells with LPS alone resulted in significant increases in cytokine production as compared to the control group (p < 0.001). However, the expression of TNF-α in the EA325E 100 µg/mL treated group showed a significant decrease as compared to the LPS group (p < 0.001). With physalin 1-100 µg/mL treatment, there was a markedly decrease in the mRNA expression levels of TNF-α in LPS stimulated raw 264.7 (p < 0.05). As shown in Figure 2B, treatment of RAW 264.7 cells with LPS alone resulted in significant increases in cytokine production as compared to the control group (p < 0.001). However, with isophysalin B 100 µg/mL treatment, there was a significant decrease in the mRNA expression levels of TNF-α in LPS-stimulated raw 264.7 cells (p < 0.001). However, there was no significant difference in the mRNA level of IL-1β after treatment of isophysalin B in LPS. As shown in Figure 2C, treatment of RAW 264.7 cells with LPS alone resulted in significant increases in cytokine production as compared to the control group (p < 0.001). The expression of TNF-α mRNA levels in physalin B 1-100 µg/mL treated group showed significantly decreased as compared to the LPS group (p < 0.05). However, there was no significant difference in the mRNA level of IL-1β after treatment of physalin B among groups. As shown in Figure 2D, treatment of RAW 264.7 cells with LPS alone resulted in significant increases in TNF-α and IL-1β mRNA expression as compared to the control group (p < 0.001). However, there was a significant difference in the mRNA level of IL-1β and TNF-α after treatment of physalin B (100 µg/mL) compared to the LPS group (p < 0.001).

Effects of Isolated Compounds from Hexane Extract in Lipopolysaccharide (LPS) Stimulated RAW 264.7 Cells
EA325H is the hexane fraction, separated from MeOH extracts of P. alkekengi var. franchetii, and a fraction was subjected to silica gel eluted with hexane-acetone, yielding 3 ,7-dimethylquercetin (rhamnazin, Figure 3A,B). The EtOAc fraction, separated by silica gel CC eluted with gradient mixtures of MeOH in CH 2 Cl 2 , was subjected to silica gel CC eluted with gradient mixtures of MeOH in CHCl3, yielding physalin B and isophysalin B. As shown in Figure 3A, treatment of RAW 264.7 cells with LPS alone resulted in significant increases in pro-inflammatory cytokines (TNF-α and IL-1β) and mRNA levels as compared to the control group (p < 0.001). The expression of TNF-α in the EA325H 1-100 µg/mL treated group showed significantly decreased as compared to the LPS group (p < 0.001).
As shown in Figure 3B, treatment of RAW 264.7 cells with LPS alone resulted in significant increases in pro-inflammatory cytokines (TNF-α and IL-1β) and mRNA levels as compared to the control group (p < 0.001). However, the expression of TNF-α mRNA levels in 3 ,7-dimethylquercetin 1-100 µg/mL treated group showed a dose-dependent decrease as compared to the LPS group (p < 0.05). Also, the expression of IL-1β mRNA levels in 3 ,7-dimethylquercetin 1-100 µg/mL treated group showed a significant decrease after 3 ,7-dimethylquercetin treatment (p < 0.001).
As shown in Figure 3C, treatment of RAW 264.7 cells with LPS alone resulted in significant increases in pro-inflammatory cytokines (TNF-α and IL-1β) mRNA levels as compared to the control group (p < 0.001). However, the expression of TNF-α mRNA levels in 3 ,7-dimethylquercetin 10 and 100 µg/mL treated group showed a significant decrease after 3 ,7-dimethylquercetin treatment (p < 0.01). Also, the expression of IL-1β mRNA levels in 3 ,7-dimethylquercetin 10-100 µg/mL treated group showed a dose-dependent decrease as compared to the LPS group (p < 0.05). These results showed that 3 ,7-dimethylquercetin can inhibit LPS-induced inflammation response in RAW264.7 cells. Data represent means ± SEM. + p < 0.05, +++ p < 0.001 compared to control, ** p < 0 *** p < 0.001 compared to LPS. Treatment of RAW 264.7 cells with LPS alone resulted in signific increases in pro-inflammatory cytokines (TNF-α and IL-1β) and mRNA levels as compared to control group. However, the expression of TNF-α and IL-1β mRNA levels in EA325H (A) and 3 dimethylquercetin (B) treated group showed a dose-dependent decrease as compared to the L group. (C) mRNA expression was determined with q-PCR.

Discussion
In this study, we demonstrated the anti-inflammatory effects of three isolated co pounds from Physalis alkekengi var. franchetii on the activation of Raw 264.7 macrophag The expression of TNF-α mRNA levels in physalin B and isophysalin 1-100 μg/mL treat group was significantly decreased as compared to the LPS group. Importantly, treatme of RAW 264.7 cells with LPS alone resulted in significant increases in pro-inflammato cytokines (TNF-α and IL-1β) and mRNA levels as compared to the control group Ho ever, the expression of TNF-α and IL-1β mRNA levels in 3′,7-dimethylquercetin 1-1 μg/mL treated group dose-dependently decreased as compared to the LPS group. The findings suggest that three isolated compounds from Physalis alkekengi var. franchetii c strongly suppress the inflammatory response to LPS in macrophages.
Physalins possess an unusual steroidal ring skeleton. Physalins were isolated fro physalis species such as physalis angulate, Physalis alkekengi var. franchetii and Physa lancifolia. Interestingly, these steroids demonstrated diverse pharmacological activiti Figure 3. Effects of isolated compounds from hexane extract in lipopolysaccharide (LPS) stimulated RAW 264.7 cells. Data represent means ± SEM. + p < 0.05, +++ p < 0.001 compared to control, ** p < 0.01, *** p < 0.001 compared to LPS. Treatment of RAW 264.7 cells with LPS alone resulted in significant increases in pro-inflammatory cytokines (TNF-α and IL-1β) and mRNA levels as compared to the control group. However, the expression of TNF-α and IL-1β mRNA levels in EA325H (A) and 3 ,7-dimethylquercetin (B) treated group showed a dose-dependent decrease as compared to the LPS group. (C) mRNA expression was determined with q-PCR.

Discussion
In this study, we demonstrated the anti-inflammatory effects of three isolated compounds from Physalis alkekengi var. franchetii on the activation of Raw 264.7 macrophages. The expression of TNF-α mRNA levels in physalin B and isophysalin 1-100 µg/mL treated group was significantly decreased as compared to the LPS group. Importantly, treatment of RAW 264.7 cells with LPS alone resulted in significant increases in pro-inflammatory cytokines (TNF-α and IL-1β) and mRNA levels as compared to the control group However, the expression of TNF-α and IL-1β mRNA levels in 3 ,7-dimethylquercetin 1-100 µg/mL treated group dose-dependently decreased as compared to the LPS group. These findings suggest that three isolated compounds from Physalis alkekengi var. franchetii can strongly suppress the inflammatory response to LPS in macrophages.
Physalins possess an unusual steroidal ring skeleton. Physalins were isolated from physalis species such as physalis angulate, Physalis alkekengi var. franchetii and Physalis lancifolia. Interestingly, these steroids demonstrated diverse pharmacological activities. One of the physalins, physalin B from Physalis angulata L. (Solanaceae) is occurring secosteroid with anti-inflammatory activities and antibacterial effects [1,[6][7][8][9]. A preclinical study reported that the physalin B inhibits the human HC116 colon cancer cell line viability [10]. Physalin B and physalin F inhibited the growth of several human leukemia cells [11]. Also, isophysalin showed high antibacterial activities against Escherichia coli and Bacillus subtilis [12]. Another study reported that Physagulin A, physagulin C, and physagulin H could not only inhibit the release of NO, PGE 2 , IL-6, and TNF-α [13].
As shown in Figure 2C, treatment of RAW 264.7 cells with LPS alone resulted in significant increases in cytokine production as compared to the control group. The expression of TNF-α mRNA levels in physalin B 1-100 µg/mL treated group was significantly decreased as compared to the LPS group. However, there was no significant difference in the mRNA level of IL-1β after treatment of physalin B among groups. To further confirm the antiinflammatory activity of physalin B, the qPCR experiments were performed. As shown in Figure 2D, treatment of RAW 264.7 cells with LPS alone resulted in significant increases in TNF-α and IL-1β mRNA expression as compared to the control group. However, IL-1β mRNA expression was markedly decreased after treatment with physalin B, which were inconsistent results with PCR results. The different results of IL-1β mRNA expression between traditional PCR and qPCR experiments were observed in the present study. The reasons for this difference are not known, but procedural or methodological differences between traditional PCR and qPCR experiments such as primer degradation, contamination, or sample variations might be responsible for these differential results. We observed that there were no significant changes in IL-1β mRNA expression after treatment with physalin B in the PCR experiment. As well known, PCR is relatively a simple qualitative technique and allows only reading the result as presence or absence of expression of IL1β mRNA levels. Even though RT-PCR is a sensitive method for the detection of low-abundance mRNA, there were reported substantial problems associated with its true sensitivity, and specificity inherent in PCR. The observed IL1 β mRNA results from our RT-PCT study may be a non-specific response since this technique compromises the specificity of the reaction. Therefore, it may be unable to distinguish real low-level transcription from false-positive transcription arising from amplification of contaminating genomic DNA with this PCR experiment. In contrast, we found that IL-1β mRNA expression was markedly decreased after treatment with physalin B as seen in Figure 2D in the qPCR as a quantitative technique, providing more reliable and reproducible quantification of IL-1β mRNA. Based on our results of qPCR, we concluded that physalin B at 100 µg/mL completely abolished LPS-induced TNF-α and IL-1β mRNA expression in Raw 264.7 cells, suggesting that the methanol extract of PAF can prevent LPS-induced inflammatory response through the downregulation of IL-1β and TNF-α in RAW 264.7 macrophages. Therefore, it could be possible to consider PAF as a potential therapeutic agent.

Conclusions
In this study, for the first time, our results demonstrated the anti-inflammatory effects of three isolated compounds from Physalis alkekengi var. franchetii on the activation of Raw 264.7 macrophages. The expression of TNF-α mRNA levels in physalin B and isophysalin B 1-100 µg/mL treated group was significantly decreased as compared to the LPS group. Importantly, treatment of RAW 264.7 cells with LPS alone resulted in significant increases in pro-inflammatory cytokines (TNF-α and IL-1β) and mRNA levels as compared to the control group. However, the expression of TNF-α and IL-1β mRNA levels in 3 ,7dimethylquercetin 1-100 µg/mL treated group dose-dependently decreased as compared to the LPS group. These findings suggest that three isolated compounds from Physalis alkekengi var. franchetii can strongly suppress the inflammatory response to LPS in macrophages.